The recent discovery of mammalian homologues of the Drosophila Toll receptor protein has elicited interest in defining the role of these proteins in innate immunity. The related Drosophila proteins Toll and 18-Wheeler are required for ani-fungal and anti-bacterial responses in the fly. The human genome encodes at least 10 distinct Toll-like receptor (TLR) protein genes, although their functions in vivo are largely unknown. It has been proposed that macrophages are likely to utilize TLR proteins as part of their responses against bacterial pathogens. In mice, the mutation in a single TLR gene results in a loss of responsiveness to Gram-negative bacterial lipopolysaccharide (LPS), and increased susceptibility to bacterial infection. We, and others, have recently characterized the TLR-dependent activation of macrophages by whole bacteria and several purified bacterial cell wall components. These studies have shown that distinct bacterial products activate cells via different TLR proteins and require CD14, an LPS receptor that also binds a variety of bacterial cell wall components. Unlike these many bacteria, TLR-dependent activation of cells by Mycobacterium tuberculosis bacilli does not require CD14. Mycobacterial ligands responsible for cellular activation by distinct TLR- dependent pathways have been identified, but have not been biochemically characterized. The long-term objectives of this project are to characterize TLR-dependent signaling, and to define the roles that these signals play in eliciting innate immune responses in macrophages. The aims of this proposal are to (1) determine whether engagement of different TLR proteins activates both shared and distinct signal transduction pathways, (2) determine if engagement of different TLR proteins activates distinct functional responses in macrophages, and (3) determine if selective loss of TLR function results in altered innate immunity.